Microcephaly (abnormally small brain) is a neurodevelopmental disorder that causes neurological symptoms, such as intellectual disability, language delay, and epilepsy. A number of causative genes have been reported, the majority of which encode centrosomal proteins. Exactly how mutations in centrosomal proteins cause microcephaly is not well understood. Previous studies using fly and mouse models suggest that mutations in centrosomal proteins may disrupt proliferation of neural progenitor cells (NPCs) or induce premature differentiation into neurons at the expense of NPCs. However, currently available animal models of microcephaly have pretty mild phenotypes, making it hard to address which molecular and cellular mechanisms are critical to severe microcephaly in humans. This project seeks to develop and establish new animal models for microcephaly with robust phenotypes. We have two hypotheses: (1) because many microcephaly gene products colocalize in the centrosome, some of them may interact biochemically and genetically. For example, ASPM (abnormal spindle-like, microcephaly-associated) and WDR62 (WD repeat domain 62), the two most common causes for human microcephaly when mutated, interact with each other. Thus, heterozygous deletion of one microcephaly gene, which has no phenotype at all by itself, may enhance the mild phenotype in homozygous knockout mice of another microcephaly gene; (2) Unlike mice, ferrets have an enlarged brain, which contains outer radial glial cells, a type of NPCs that is highly abundant in the human cortex. Thus, knockout ferrets of a microcephaly gene may show robust phenotypes compared to knockout mice of the same gene. In Aim 1, we will examine Aspm-/-; Wdr62+/- mice that have a significantly smaller brain than any control mice (Aspm+/+; Wdr62+/- and Aspm-/-; Wdr62+/+ mice), which have negligible phenotypes. We will characterize interaction between the two proteins as well as asymmetric inheritance of mother versus daughter centrosomes in the developing cortex of Aspm-/-; Wdr62+/- mice. In Aim 2, we will establish and characterize Aspm-knockout ferrets that we have recently generated using TALEN, a new genome-editing tool. Preliminary data show that they have severe microcephaly. Using immunohistochemistry and adenoviral green fluorescent protein infection followed by time-lapse imaging, we will examine abundance and behaviors of diverse NPCs in Aspm-knockout and wile-type ferrets. The proposed work will provide exciting new animal models for microcephaly and cortical malformation in general, creating an innovative experimental system in the field of cerebral cortical development and evolution. With ever-increasing list of genes from human genetic studies, our approach will demonstrate how to study functional meanings of a gene when knockout mice of the gene do not show robust phenotypes. In addition, it should resolve the current debate over roles of outer radial glial cell during cerebral cortical development, and has the potential to identify novel mechanisms of normal cortical development.